Where can I check which HLA Database is used for analysis?
In SEQUENCE Pilot the used HLA database can be viewed and changed in menu SeqHLA / HLA DB Admin….
How can I use and update NMDP codes?
NMDP codes can be generated for every result. To activate the NMDP code generation create the new subdirectory NMDPFiles in directory SeqPilot of your installation.
Within this directory the current NMDP list (numer.v3.txt) has to be deposited:
Please note, that you have to update the NMDP list regularly. Therefore please use the procedure as descibed above.
How are G and P suffixes calculated in SEQUENCE Pilot?
The G and P suffixes are used to simplify the results in operation Sequence dialogue Result. By default SEQUENCE Pilot calculates the G and P suffixes automatically.
- “G” stands for alleles which share identical nucleotide sequences for the exons encoding the peptide binding domain exon 2 and 3 for HLA class I and exon 2 only for HLA class II alleles have to be identical in nucleotide sequence, then a G Suffix is generated.
- “P” stands for alleles, which encode for identical peptide binding domains: exon 2 and 3 for HLA class I and exon 2 only for HLA class II alleles have to be identical in protein sequence, then a P Suffix is generated.
Alternatively files from IMGT can be downloaded where all G- and P-Groups are defined. In case you use this option, you have to update the files regularly.
The reporting of ambiguous HLA allele typing only works, if you use maximum resolution.
How can I use the G and P suffixes from IMGT files instead of the calculated ones from SEQUENCE Pilot?
Files from IMGT can be downloaded where all G- and P-Groups are defined. In case you use this option, you have to update the files regularly (with every database update).
To use the nomenclature from a file, please do the following:
- Go to http://hla.alleles.org/wmda/.
- Download the files hla nom g.txt and hla nom p.txt.
- Save the files in the folder of the respective database in folder Settings\HLANom of your SEQUENCE Pilot installation (e.g. the files for HLA DB 3.31 must be saved in folder Settings\HLANom\3.24.0).
- Open the lis.ini file, located in SeqPilot\bin your SEQUENCE Pilot installation with a text editor.
- Go to section [SeqPilot] and deposit the entry HLANomFileAllele=yes in a new line.
- After restarting SEQUENCE Pilot the G and P suffixes for all newly started runs will be used from this file.
How can I inactivate G and P suffixes?
In case you do not want to use G and P suffixes at all, enter the new line Enter ReportAmbigousAllele=no in the section [SeqNextHLA] of your in the lis.ini file, located in the SeqPilot\bin of your SEQUENCE Pilot installation.
Where can I find the result?
In operation Sequence, section Result. Here the result, calculated for the selected gene in the dialogue part Genes is listed.
The first line shows, whether it is a heterozygous or a hemizygous result and on which haplotypes the result calculation is based on. The second line shows the ambiguities, that is all found allele combinations. G and P suffixes are used automatically. The last line shows, which HLA database version was used for the result calculation. Optionally the result can be listed as NMDP code.
If there are still mismatches to an allele there will be the entry “no results possible”. In that case you have to check the mismatching positions in the electropherogram. When all mismatches are removed the result will appear automatically.
What does the Matching table display?
This table shows all possible hemizygous results/allele combinations, sorted by their probability.
If you select an entry within this table it is highlighted green. This allele or allele combination is displayed in the electropherogram then. If there are mismatching positions for that allele or allele combination, the affected result files are displayed automatically and the cursor is positioned to the first mismatch.
All allele combinations with no mismatches are listed as result in section Result.
Is it possible to set a preferred result in case ambiguities are present?
Yes, this is possible in the Matching table, using the context menu item prefer alleles.
Is it possible to compare the sequences of my resulting allele sequences?
Yes, this is possible in the Matching table using the context menu item show differences….
Can new alleles be called as well?
Yes, if a new allele is present one mismatch remains to all known allele combinations from the IMGT HLA database. The new allele can be set manually in operation Sequence section Genes using the context menu new allele…. Afterwards the result “new allele most similar to…” is shown in section Result.
How can I export the results?
The results shown in the Results window in operation Sequence can be exported as txt file. Therefore, please use the context menu export / result... in section Genes.
Moreover a report can be printed or saved as pdf. Therefore use the button [Print] in dialogue part Functions.
Results can also be exported automatically to any LIMS systems after analysis, with our TALKMASTER interface.
Which files can be analysed?
Fastq and fastq.gz files from all common sequencing platforms from Illumina, IonTorrent, MinION and PacBio can be analysed.
Which one time procedures need to be set up before I can start analysis?
The HLA database needs to be installed and ROIs need to be defined in operation ROI [master file].
How can I deposit the DNA number in the file name?
By default all signs in the file name before the first "_" are used as DNA number. In case this is not the DNA number of your file name, please contact our SEQUENCE Pilot support team. We can adapt our software, so that the DNA number is read correctly.
Is it possible to automatically start a Run?
Yes, with the Autorun option. For further details, please contact our SEQUENCE Pilot support team.
How can I make analysis faster?
- Switch off Second Alignment: The analysis will be faster, by adding the entry SecondLevelCalcAlignmentScore=no in the file lis.ini (SeqPilot\bin of your SEQUENCE Pilot installation) in section [SeqNextHLA]. When this entry is set a second alignment for reads which can be mapped to more than one locus is not done any more. But please note, that this might lead to single reads getting mapped to the wrong locus (especially for loci B/C or DRB pseudogenes) - depending on the allele combinations.
- Activate muliple processing cores: To make analysis faster multiple processing cores can be used to compute the result files of a run. Several worker processes can be started, which process several files in parallel (in case the Run is started for several files, each worker processes one file). The number of worker processes should be related to the number of cores available on the server / client PC. For further details please contact our SEQUENCE Pilot support team.
- Analysis is slow because the Coverage is very high: The following settings in operation Run, utton [Settings], tab Expert Settings can be adapted:
- Load file: For very big files, only part of the reads can be loaded and analysed. This makes analysis much quicker. Here the percentage of reads that should be loaded and used for analysis can be entered. 100% means, that all reads are loaded.
- Load reads: Here an absolute minimum number of reads that must be loaded can be entered. This setting can overrule the setting Load file mentioned above. In case the percentage value is too low to load the number of reads defined here, more reads are loaded until the number of reads defined.
Analysis is slowing down my PC. What can I do?
By default the importer process uses the maximum number of cores. Therefore the computer might be very slow during data analysis, which might be a problem in case other programs are used as well.
The number of cores used during import can be restricted with the entry MaxImporterThreadCores=1 in section [SeqNextHLA] the lis.ini file (SeqPilot\bin of your SEQUENCE Pilot installation). The number behind this entry is the number of cores used for import.
Can I load data for the same patient several times?
This should only be done in case the fastq files contain data for different loci. In case you want to load replicates for one patient of the same loci, please contact our SEQUENCE Pilot support team to discuss all possible options.
Are introns analysed as well?
Intron sequences are displayed but not used for result calculation. They are only used to exclude known null alleles.
I have two possible results listed. Is it possible to exclude one of them?
Yes, phasing can optionally be checked. If possible, allele combinations are then removed from the result based on the phasing. To use this option, please activate the setting "Check phasing" on tab Expert Settings.
Can I exclude null alleles?
For result calculation null alleles which show mismatches/insertions/deletion in the introns are called or excluded as result automatically. This function is active by default, but it can be switched off. To switch it off the setting "Check null allele intron" on tab Expert Settings has to be deactivated.
How can I change the resolution?
By default 3 field resolution (maximum resolution) is used.
In case another resolution should always be used (2 field = 4 digits or 1 field= 2 digits), this can be set in the lis.ini file, located in folder SeqPilot\bin of your SEQUENCE Pilot installation. To change to 2 digits or 4 digits, please make the entry DefaultResolution=2 digits or DefaultResolution=4 digits in section [SeqNextHLA].
For already analysed files open the patient sample in operation Sequence, right click a locus in section Genes and select item change resolution and positions… from the context menu.
What can I check in case a result is not called automatically?
Please contact our SEQUENCE Pilot support team.
What can I do in case background is present for one or several loci?
It needs to be checked to which locus the background belongs to. In case it belongs to a locus which is not set up as ROI (e.g. locus E, G, Y…), ROIs for this locus need to be set up in ROI [master file] and included in the corresponding ROI group (ROI Group [master file]). In case you do not want to call results for these ROIs, you can set them to analysis mode “only mapping” in ROI [master file].
A result is called but mismatches are shown for the locus. What does this mean?
A result is always called as soon as there are no mismatches in exon areas. In intron areas mismatches are not regarded for result calculation.
In operation Sequence allele sequences from the IMGT HLA database are marked blue instead of green. What does this mean?
In case an intron sequence is unknown for a haplotype, it is checked if the intron sequence of another group member is known.
If yes, the sequence of the group member is listed. The haplotype sequence for the intron is then marked blue instead of green. You can see which allele the intron sequence belongs to when you move your mouse to the blue intron sequence (tool tip). This feature can be switched off using the entry Show2DigitsIntronAlleles=no in section [SeqPilot] of the lis.ini file (SeqPilot\bin of your SEQUENCE Pilot installation).
If no, no intron sequence is shown.
In operation Sequence the number in front of the read sequences can be highlighted in different colors. Moreover a * can be in front. What does this mean?
Identical reads are listed only once. The copy number is listed in front of each read. Moreover all reads that have a perfect match to an allele can optionally be marked with a * in front of the number.
The * and the copy number in front of each read can be colored. Identical colors for two or more reads mean, that the reads have a perfect match to the same allele(s) (context menu item "show alleles lists identical matches").
Please note, that black is not regarded as a color. Black */copy numbers mean that no other reads with the same perfect matches are present.
Can I get a quick overview about coverages?
Yes, therefore please press the button [Summary] in operation Sequence. The table can be exported as txt-file using the corresponding context menu item.